Figure 4. Reduction in PfRhopH2 expression leads to altered growth phenotypes in vitro.
(a) Effect of glucosamine on PfRhopH2 protein expression. Upper panel: overview of experiment. Synchronised cultures of PfRhopH2-glmS were treated with glucosamine (GlcN) at the indicated time and material harvested, as indicated. Lower panels: infected erythrocytes were harvested by saponin lysis and subject to SDS-PAGE and western blotting. PfRhopH2-HA was detected using an anti-HA antibody and EXP2 (used as a loading control) detected with a specific polyclonal EXP2 antibody. Right panel: Densitometry performed on bands observed in western blot using ImageJ was performed to calculate the ratio of EXP2 or RhopH2 protein levels in parasite lines grown in the presence (+) or absence (-) of GlcN (n = 3 independent experiments). Shown is the mean ± SEM (n = 3). (b) Representative Giemsa-stained smears parasites depleted of RhopH2 progress to schizont stage in cycle one but parasite growth is slowed around the trophozoite stage (n = 3 independent experiments). (c) Analysis of the number of schizonts in cultures of wildtype (3D7) and RhopH2-HAglmS parasites grown in the absence (−) or presence (+) of 2.5 mM GlcN that invaded donor erythrocytes within 3 or 5 hr post-incubation (hpi), as measured by FACS (n = 3). Shown is the mean ± SEM. (d) Box plot indicating the number of merozoites formed per schizont in cultures of RhopH2-HAglmS grown in 0 mM (35 schizonts examined) or 2.5 mM (51 schizonts examined) GlcN. The central bar in the box plot denotes the median whilst the whiskers delineate the 10th and 90th percentiles. p<0.0001 by unpaired t-test. (e) Parasitemias of cultured PfRhopH2-HAglmS parasites grown in 0 mM or 2.5 mM GlcN, determined by counting a minimum of 1000 erythrocytes. Depletion of PfRhopH2 expression increases the length of the cell cycle and has a marked effect on the numbers of parasites progressing to cycle 3. Shown is the mean ± SEM (n = 3). (f) Growth of 3D7 and PfRhopH2-HAglmS parasites when cultured in various concentrations of GlcN, as measured by lactate dehydrogenase assay (LDH). The LDH activities of 3D7 and RhopH2-HAglmS cultured in the absence of GlcN at cycle three were normalized to 100%, and activity of all lines (± GlcN) across the three cycles was measured relative to this. Shown is the mean ± SD (n = 3). An unpaired t-test revealed RhopH2-HAglmS parasites grew significantly slower than 3D7 in all concentrations of GlcN by 36 hpi (p<0.01) (g) Measurement of nanoluciferase (Nluc) released into the culture media and in pelleted erythrocytes infected with 3D7 or RhopH2-HAglmS parasites expressing Hyp1-Nluc. Measurements commenced around the time 3D7 parasites were starting to egress and invade new erythrocytes. The data represents the mean ± SD of one biological replicate completed in triplicate, with results expressed as percentage Nluc activity in the media relative to the pellet fraction.